The present invention relates to a method for delivering an agent to a target site.
The delivery of a therapeutic agents to specific tissues is desirable typically to ensure that a sufficiently high dose of a given agent is delivered to a selected tissue. Moreover, it is often the case that the therapeutic agent, although advantageously having beneficial therapeutic effects on the diseased tissue, may have undesirable side effects on tissues that are not diseased. For example, in the treatment of certain types of disorders, such as cancer, it is necessary to use a high enough dose of a drug to kill the cancer cells without killing an unacceptable high number of normal cells. Thus, one of the major challenges of disease treatment is to identify ways of exploiting cellular drug delivery vehicles to incorporate and to selectively release agents at a desired target site.
It has been suggested that red blood cells may be exploited as active agent/drug delivery vehicles (DeLoach and Sprandel 1985, Bibliotheca Haematologica; Publ. Karger, Munich) as it is possible to incorporate agents into human red blood cells using a variety of techniques. An example of such a technique is the exploitation of osmotic shock and modifications thereof such as hypotonic shock and subsequent recovery of isotonicity and reverse hypotonic dialysis (Luque and Pinilla, 1993, Ind. Farmac. 8, 53-59).
An alternative method for loading drugs and active agents into red blood cells is electroporation. Using this process, the agent of interest are mixed with the live red blood cells in a buffer medium and short pulses of high electric fields are applied. The red blood cell membranes are transiently made porous and the agents of interest enter the cells. The electroporation process is advantageous as very high loading indices can be achieved within a very short time period (Flynn et al., 1994, Cancer Letts., 82, 225-229).
When packaging/carrier/delivery systems such as red blood cells are used as in vivo delivery systems, they suffer from the drawback that the delivery function is dependent upon both an accumulation of the red blood cells and a breakdown of the red blood cell membrane in or at the relevant tissue/site. As a result, attempts have been made to incorporate sensitizing agents into cell carriers in order to facilitate both the accumulation and/or release of an agent of interest at a target site.
Alternative energy sources have been investigated as tools for inducing payload release from loaded and sensitized cells. By way of example, ultrasound irradiation has been investigated as an alternative to light induced photodynamic activation as it has a broader degree of focus and it penetrates more deeply into the body. However, although ultrasound irradiation has also been applied to effect red blood cell lysis in vitro, its use has been limited in that its effect is only significant at lower cell concentrations (1-6xc3x97106 cells) (Brayman et at., 1996, Ultrasound in Med and Biol., 22: 497-514). Moreover, ultrasound is non-specific in effects, resulting in lysis of both loaded and endogenous red blood cells.
Recently, it has been found that certain dye compounds, in particular porphyrins, can achieve a cytopathogenic effect when the disease site is subjected to ultrasound irradiation. This technique is referred to as sonodynamic therapy and is discussed in WO98/52609. WO98/52609 teaches that ultrasound irradiation may be useful in treating disease but only when it is combined with an effective amount of an ultrasound-susceptibility modification agent such as a porphyrin.
The present invention provides a method for selectively releasing an agent from a loaded red blood cell at a target site.
According to a first aspect of the present invention, we provide the use of an electric field for sensitizing a red blood cell to ultrasound.
Preferably, the electric field is used in a method which comprises the steps of: providing a red blood cell and subjecting the red blood cell to an electric field, the electric field having sufficient energy to electrosensitize the cell. More preferably, the red blood cell sensitized using electric field pulsing may be selectively disrupted using ultrasound.
According to a second aspect of the invention, we provide a method of selectively disrupting a red blood cell, the method comprising the steps of: (a) providing a red blood cell; (b) electrosensitizing said red blood cell; and (c) disrupting said red blood cell by subjecting said red blood cell to ultrasound.
Preferably, the use according to the first aspect of the invention and the method according to the second aspect of the invention is such that the electrosensitization comprises the step of applying an electric pulse to a red blood cell. Preferably, the electric pulse is from about 0.1 kVolts/cm to about 10 kVolts/cm under in vitro conditions.
The method or use according to the first and second aspects of the invention may further comprise the step of loading the red blood cell with an agent.
The sensitization of the red blood cell may precede the loading of the agent. Alternatively, the loading of the agent precedes the sensitization of the red blood cell. In yet another alternative, the sensitization of the red blood cell and the loading of the agent are substantially simultaneous.
According to a third aspect of the invention, we provide a method for selectively releasing an agent from a red blood cell comprising the steps of: loading a red blood cell with an agent; electrosensitizing the red blood cell; and causing the agent to be released from the electrosensitized red blood cell by applying ultrasound at a frequency and energy sufficient to cause disruption of the red blood cell but insufficient to cause disruption of unsensitized red blood cells.
According to a fourth aspect of the present invention, there is provided a method for delivering an agent to a target site in a vertebrate, comprising the steps of: providing a red blood cell; loading the red blood cell with an agent; electrosensitizing the red blood cell; introducing the sensitized red blood cell into the vertebrate; and causing the disruption of the sensitized red blood cell by treatment of the cell with ultrasound to release the agent at a target site.
According to a fifth aspect of the present invention, there is provided a method for electrosensitizing a red blood cell, comprising the steps of: providing a red blood cell; and subjecting the red blood cell to an electric field, the electric field having sufficient energy to electrosensitize the cell.
The electrosensitized red blood cells according to the invention may be loaded with agents either before, during or after the electrosensitization procedure. In one aspect, therefore, the electrosensitization procedure is effective to electroporate the cells, thus effecting simultaneous loading of a desired agent. Preferably, however, the electrosensitization procedure is not effective to electroporate the cells, and the loading is thus carried out in a separate step either before or after the electrosensitization procedure. Preferred methods for loading cells are set out below.
According to a sixth aspect of the invention, there is provided an electrosensitized red blood cell which is preparable by subjecting a red blood cell to an electric field at an energy level which is not effective to electroporate the cell. The invention also provides electrosensitized red blood cells according to the fourth aspect, which have been loaded with an agent using a process other than electroporation.
According to a seventh aspect of the present invention, there is provided a kit comprising a red blood cell, an agent, packaging materials therefor and instructions for use, the use comprising the steps of: electrosensitizing a red blood; loading the red blood cell with an agent; causing the agent to be released from the electrosensitized red blood cell by exposure to ultrasound at a frequency and energy effective to cause disruption of the sensitized red blood cell but insufficient to cause disruption of unsensitized red blood cells.
According to a eighth aspect of the present invention, there is provided a kit comprising a red blood cell which is loaded with an agent, packaging materials therefor and instructions for use comprising the steps of: electrosensitizing a red blood cell; and causing the agent to be released from the sensitized red blood cell by exposure to ultrasound at a frequency and energy effective to cause disruption of the sensitized red blood cell but insufficient to cause disruption of unsensitized red blood cells.
According to a ninth aspect of the present invention, there is provided a kit comprising a loaded electrosensitized red blood and instructions for causing the agent to be released from the electrosensitized red blood cell by exposure to ultrasound at a frequency and energy effective to cause disruption of the sensitized red blood cell but insufficient to cause disruption of unsensitized red blood cells.
In these and other aspects of the invention, the sensitization and loading steps may be performed in any desired order, as appropriate.